The long-term objective of this work is to understand the interactions which are important for protein structure and function. Protein folding, the acquisition of a final, three-dimensional tertiary structure from a primary amino acid sequence, is the final step in translating the genetic message encoded in the DNA into a functional product. This tertiary structure is very specific: the mutation of a single amino acid residue in a single protein can cause transformation or eliminate viability of an otherwise healthy cell. Many different technologies are being used to study this problem. Site-directed mutagenesis has been used to determine the importance of specific residues for the folding or function of a specific protein. Directed, random mutagenesis, one form of site-directed mutagenesis, is a technique in which a residue or residues are mutated into all possible amino acids In a single experiment, which allows much more Information about a residue's "replaceability" to be determined at one time. In another form of mutagenesis, oligonucleotide-directed mutagenesis, whole protein segments - secondary structures, supersecondary structures, or domains - are modified with a single oligonucleotide. The degree of hierarchical structure or modularity present In a protein can be determined from this type of experiment. The research described in this proposal will apply these techniques - directed, random mutagenesis and oligonucleotide-directed mutagenesis - to iso-l-cytochrome c, an important and highly conserved electron transport protein in the yeast Saccharomyces cerevisiae, In order to determine the affect or the mutations on the structure and function of this protein. Oligonucleotide-directed mutagenesis is already in use in this system, but the techniques of random mutagenesis must be first developed. This work, then, has four specific goals: 1) To continue oligonucleotide-directed mutagenesis of loops and helices In Iso-l-cytochrome c to determine which regions are structurally or functionally important; 2) To study the spectroscopic and kinetic properties or the mutant proteins in vivo and in vitro to determine how each mutation affects the protein structure and function; 3) To develop a system for performing directed, random mutagenesis in Iso-l-cytochrome c; and 4) To use random mutagenesis to further probe structure and function relationships in this protein. The results of directed, random mutagenesis and oligonucleotide-directed mutagenesis have not before been consistently applied in this system, but will lead to a better understanding of the specific residues or groups of residues involved in the folding and function of cytochrome c; furthermore, any general folding rules deduced for cytochrome c may be applicable to the folding of other proteins.